The broad goal of this research program is to describe the network of regulatory elements that control the initiation of DNA replication and couple it to the cell cycle, with particular focus on a conserved family of molecules called MCM proteins. These proteins are found in eukaryotes ranging from yeast to man. Mutations in MCM proteins impair DNA replication, resulting in chromosome damage and genetic instability. Such defects in genome integrity can cause or contribute to development of cancer, and can also cause defects in development. Thus, understanding the regulation of DNA replication has direct relevance to human health. This proposal exploits the genetics of a model eukaryotic system, the fission yeast S. pombe, to investigate the role of the MCM proteins in DNA replication. Eukaryotic cells contain six related MCM proteins assembled in a large complex, and each protein is essential for viability. What distinguishes these six proteins from one another? What functions does each provide to the assembly, localization, and activity of the MCM complex? The fission yeast system is particularly well suited to address these questions, because the structure of its replication origins and behavior of its MCM proteins closely resemble those in higher eukaryotes, and it is easily manipulated using genetics and molecular biology. In order to identify the contribution of each MCM protein to complex assembly and localization in s. pombe, individual MCMs will be subjected to mutational analysis and the effects of these mutations upon complex behavior in vivo will be examined using established protocols. Effects of mcm mutations on DNA replication and cell cycle progression will be determined. Genetic and physical interactions with known genes will be investigated, and evidence for interactions between MCM proteins and known cell cycle regulators will be sought. Finally, a genetic screen will be employed to identify additional genes with which the MCM proteins interact. Throughout the study, the behavior of individual MCMs, their effect on replication, and their position within the MCM complex will be correlated. This study will contribute to a complete description of how these conserved proteins are assembled and function within the network of proteins inside the living cell.